The kappaE3' enhancer is a strong, B cell specific enhancer that lies 8.5 kilobases downstream of the immunoglobulin kappa constant region exon. The activity of this enhancer is controlled during B cell development. That is, it is silent at the pre-B cell stage, but active at the B cell and plasma cell stages. Our goals are to identify and characterize the DNA sequences and trans-acting factors that are responsible for the developmental control of kappaE3' enhancer activity. We will functionally characterize 3 proteins (PU.1, NF-EM5, and NF-E1) that bind to kappaE3' enhancer sequences. PU.1 is an ets-related transcription factor that binds to the kappaE3' enhancer and recruits the binding of a second B cell-specific factor NF-EM5. Binding of both factors appears to be important for kappaE3' enhancer activity. We will characterize the interaction between PU.1 and NF-EM5 by several approaches. First, we will prepare specific mutations in the PU.1 sequence and determine the ability of the mutant proteins to interact with NF-EM5. Second, we will determine whether the phosphorylation status of PU.1 is controlled during B cell development. The protein-protein interaction between these proteins requires phosphorylation of PU.1 and this may provide a mechanism for the developmental control of enhancer activity. Finally, we will characterize candidate NF-EM5 cDNA clones that encode proteins that physically interact with PU.1. The third protein that we will characterize, NF-E1, binds to a negative-acting region of the kappaE3' enhancer. This factor can either repress or activate transcription depending upon promoter context or cellular concentration. This dual function of NF-E1 may relate to the developmental control of kappaE3' enhancer activity. We will prepare mutations in the NF-E1 sequence to identify the domains responsible for transcriptional activation and repression. We will then perform experiments designed to detect proteins that interact with NF-E1 and control its activity. Ultimately, understanding the developmental control of kappaE3' enhancer activity requires understanding the function of each enhancer motif. We have determined that the kappaE3' enhancer is composed of both positive- as well as negative-acting DNA sequences. We will determine the role of each positive-acting enhancer motif at each stage of B cell development by preparing site-specific mutations in each motif. Constructs will be assayed for enhancer activity after transfection into cell lines representative of each stage of B cell development. We will also study the mechanism of action of the negative-acting sequences that are responsible for silencer activity at the pre-B cell stage. We will determine whether these DNA sequences can repress heterologous transcriptional control elements and whether their function is B cell specific. We will also attempt to identify the specific targets of silencer activity. The above experiments will enable us to determine the relative importance of each enhancer motif at each stage of B cell development. Our long-term goals are to determine whether the factors that control kappaE3' enhancer activity also play a role in B cell differentiation.